It has been recognized for decades that solid tumors can contain regions at very low oxygen concentrations (hypoxia) which do not occur in normal tissues under physiological conditions. The central focus of this Program Project Grant is on this tumor hypoxia. The overall goal is to develop means to exploit in cancer treatment this key difference between tumors and normal tissues. Approximately eight years ago, we discovered a novel compound, SR 4233, which has very high selective cytotoxicity for hypoxic cells. When given appropriately with radiation or with chemotherapeutic drugs, this compound, now known as tirapazamine, can exploit the hypoxia in tumors, thereby rendering them even more sensitive to treatment than if the tumors had no hypoxic cells. This drug is now about to enter Phase II and III clinical trials. However, appropriate patient selection will be key to their success, and the first three projects in this proposal relate to various aspects of this question. The two principal questions that will be asked, with human tumor xenografts and in a clinical trial with head and neck cancers, will be first, whether the response of tumors to the addition of tirapazamine to fractionated radiation can be predicted, both from their level of hypoxia and from their cellular content of bioreductive enzymes; and second, whether the increased response of the tumors can be measured during the treatment using a surrogate, or intermediate, endpoint. Of key importance in these studies will be the appropriate measurements of tumor oxygenation, and we will investigate this with animal tumors and human tumors to determine which assay for tumor hypoxia is most accurate and most convenient for use in the clinical setting. Increasingly, hypoxia in tumors is seen as a cellular stress that can have a profound impact on the biology and progression of the tumor. Several years ago, novel proteins were identified as arising in hypoxic cells, and the last two projects focus on the cellular and molecular consequences of these proteins induced by hypoxia and/or reoxygenation of the tumor cells. These studies will be performed with cells in vitro, with cells in multicellular spheroids, and with animal and human tumors. A principal goal will be to span the gap between what is presently known about genes induced under hypoxic conditions and the significance of this for human tumors, both for their progression and for their response to treatment. This project represents a highly coordinated program between an outstanding group of investigators with expertise at the molecular, cellular, animal, and clinical levels. The group is uniquely positioned to take advantage of the latest developments in this field, including use of the hypoxic, cytotoxin, tirapazamine, use of the new commercially available oxygen polarographic electrode, and the new findings on the molecular biology of cellular hypoxia.